Abstract

The ketone body acetoacetate (AA) in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by ∼2- to 2.5-fold, respectively, following 6-h treatment. The AA-mediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2′-amino-3′-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by ∼50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from ∼24 h in untreated hepatocytes to ∼32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by ∼2- and 2.5-fold at 24 and 48 h, respectively. dl-β-Hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by ∼40%. CYP2E1 protein half-life increased from ∼8 h in untreated hepatocytes to ∼24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.